AZIN1 RNA editing alters protein interactions, leading to nuclear translocation and worse outcomes in prostate cancer.

The transcript encoding Antizyme Inhibitor 1 (AZIN1) is frequently edited in various cancers, and this editing is associated with enhanced tumor aggressiveness. After comparison of wild-type AZIN1 (wtAZIN1) and edited AZIN1 (edAZIN1, which contains a Ser367Gly substitution), we report differential binding of edAZIN1 to a small set of proteins; specifically, edAZIN1 binds to alpha-smooth muscle actin (ACTA2), gamma actin 1 (ACTG1), and myosin9, whereas wtAZIN1 does not. This binding enables nuclear translocation of edAZIN1. In contrast to overexpression of edAZIN1 and, to a lesser extent, (editable) wtAZIN1, overexpression of an uneditable AZIN1 allele does not promote a cellular phenotype associated with increased tumorigenicity. In patients, both editing and nuclear localization of AZIN1 are common and are associated with tumor aggressiveness, i.e., a higher Gleason score, higher genomic instability, and a shorter progression-free survival time. In conclusion, the data indicate that binding of edAZIN1 to the actin/myosin9 complex supports its nuclear translocation, leading to enhanced cellular aggressiveness, and is associated with worse prostate cancer outcomes.

Adjuvant-pulsed mRNA vaccine nanoparticle for immunoprophylactic and therapeutic tumor suppression in mice.

Synthetic mRNA represents an exciting cancer vaccine technology for the implementation of effective cancer immunotherapy. However, inefficient in vivo mRNA delivery along with a requirement for immune co-stimulation present major hurdles to achieving anti-tumor therapeutic efficacy. Here, we demonstrate a proof-of-concept adjuvant-pulsed mRNA vaccine nanoparticle (NP) that is composed of an ovalbumin-coded mRNA and a palmitic acid-modified TLR7/8 agonist R848 (C16-R848), coated with a lipid-polyethylene glycol (lipid-PEG) shell. This mRNA vaccine NP formulation retained the adjuvant activity of encapsulated C16-R848 and markedly improved the transfection efficacy of the mRNA (>95%) and subsequent MHC class I presentation of OVA mRNA derived antigen in antigen-presenting cells. The C16-R848 adjuvant-pulsed mRNA vaccine NP approach induced an effective adaptive immune response by significantly improving the expansion of OVA-specific CD8+ T cells and infiltration of these cells into the tumor bed in vivo, relative to the mRNA vaccine NP without adjuvant. The approach led to an effective anti-tumor immunity against OVA expressing syngeneic allograft mouse models of lymphoma and prostate cancer, resulting in a significant prevention of tumor growth when the vaccine was given before tumor engraftment (84% reduction vs. control) and suppression of tumor growth when given post engraftment (60% reduction vs. control). Our findings indicate that C16-R848 adjuvant pulsation to mRNA vaccine NP is a rational design strategy to increase the effectiveness of synthetic mRNA vaccines for cancer immunotherapy.

Lipidation Approaches Potentiate Adjuvant-Pulsed Immune Surveillance: A Design Rationale for Cancer Nanovaccine.

Adjuvant-pulsed peptide vaccines hold great promise for the prevention and treatment of different diseases including cancer. However, it has been difficult to maximize vaccine efficacy due to numerous obstacles including the unfavorable tolerability profile of adjuvants, instability of peptide antigens, limited cellular uptake, and fast diffusion from the injection site, as well as systemic adverse effects. Here we describe a robust lipidation approach for effective nanoparticle co-delivery of low-molecular weight immunomodulators (TLR7/8 agonists) and peptides (SIINFEKL) with a potent in vivo prophylactic effect. The lipidation approaches (C16-R848 and C16-SIINFEKL) increased their hydrophobicity that is intended not only to improve drug encapsulation efficiency but also to facilitate the membrane association, intracellular trafficking, and subcellular localization. The polymer-lipid hybrid nanoparticles (PLNs) are designed to sustain antigen/adjuvant levels with less systemic exposure. Our results demonstrated that a lipidated nanovaccine can induce effective immunity by enhancing the expansion and activation of antigen-specific CD8+ T cells. This adaptive immune response led to substantial tumor suppression with improved overall survival in a prophylactic setting. Our new methodology enhances the potential of nanovaccines for anti-tumor therapy.

Unbiased Phenotype-Based Screen Identifies Therapeutic Agents Selective for Metastatic Prostate Cancer.

In American men, prostate cancer is the second leading cause of cancer-related death. Dissemination of prostate cancer cells to distant organs significantly worsens patients' prognosis, and currently there are no effective treatment options that can cure advanced-stage prostate cancer. In an effort to identify compounds selective for metastatic prostate cancer cells over benign prostate cancer cells or normal prostate epithelial cells, we applied a phenotype-based in vitro drug screening method utilizing multiple prostate cancer cell lines to test 1,120 different compounds from a commercial drug library. Top drug candidates were then examined in multiple mouse xenograft models including subcutaneous tumor growth, experimental lung metastasis, and experimental bone metastasis assays. A subset of compounds including fenbendazole, fluspirilene, clofazimine, niclosamide, and suloctidil showed preferential cytotoxicity and apoptosis towards metastatic prostate cancer cells in vitro and in vivo. The bioavailability of the most discerning agents, especially fenbendazole and albendazole, was improved by formulating as micelles or nanoparticles. The enhanced forms of fenbendazole and albendazole significantly prolonged survival in mice bearing metastases, and albendazole-treated mice displayed significantly longer median survival times than paclitaxel-treated mice. Importantly, these drugs effectively targeted taxane-resistant tumors and bone metastases - two common clinical conditions in patients with aggressive prostate cancer. In summary, we find that metastatic prostate tumor cells differ from benign prostate tumor cells in their sensitivity to certain drug classes. Taken together, our results strongly suggest that albendazole, an anthelmintic medication, may represent a potential adjuvant or neoadjuvant to standard therapy in the treatment of disseminated prostate cancer.

Developing a novel FRET assay, targeting the binding between Antizyme-AZIN.

Antizyme inhibitor (AZIN) stimulates cell proliferation by binding to and sequestering the cell cycle suppressor antizyme. Despite the important role of the antizyme-AZIN protein-protein interaction (PPI) in cell cycle regulation, there are no assays for directly measuring the binding of AZIN to antizyme that are amenable to high throughput screening. To address this problem, we developed and validated a novel antizyme-AZIN intramolecular FRET sensor using clover and mRuby2 fluorescent proteins. By introducing alanine mutations in the AZIN protein, we used this sensor to probe the PPI for key residues governing the binding interaction. We found that like many PPIs, the energy of the antizyme-AZIN binding interaction is distributed across many amino acid residues; mutation of individual residues did not have a significant effect on disrupting the PPI. We also examined the interaction between Clover-AZIN and antizyme-mRuby2 in cells. Evidence of a direct interaction between Clover-AZIN and antizyme-mRuby2 was observed within cells, validating the use of this FRET sensor for probing intracellular antizyme-AZIN PPI. In conclusion, we have developed and optimized a FRET sensor which can be adapted for high throughput screening of either in vitro or intracellular activity.

Synthesis and anticancer activity of novel water soluble benzimidazole carbamates.

Metastases account for more than 90% of all cancer deaths and respond poorly to most therapies. There remains an urgent need for new therapeutic modalities for the treatment of advanced metastatic cancers. The benzimidazole methylcarbamate drugs, commonly used as anti-helmitics, have been suggested to have anticancer activity, but progress has been stalled by their poor water solubility and poor suitability for systemic delivery to disseminated cancers. We synthesized and characterized the anticancer activity of novel benzimidazoles containing an oxetane or an amine group to enhance solubility. Among them, the novel oxetanyl substituted compound 18 demonstrated significant cytotoxicity toward a variety of cancer cell types including prostate, lung, and ovarian cancers with strong activity toward highly aggressive cancer lines (IC50: 0.9-3.8 µM). Compound 18 achieved aqueous solubility of 361 µM. In a mouse xenograft model of a highly metastatic human prostate cancer, compound 18 (30 mg/kg) significantly inhibited the growth of established tumors (T/C: 0.36) without noticeable toxicity.

Restoration of tumour-growth suppression in vivo via systemic nanoparticle-mediated delivery of PTEN mRNA.

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a well-characterized tumour-suppressor gene that is lost or mutated in about half of metastatic castration-resistant prostate cancers and in many other human cancers. The restoration of functional PTEN as a treatment for prostate cancer has, however, proven difficult. Here, we show that PTEN messenger RNA (mRNA) can be reintroduced into PTEN-null prostate cancer cells in vitro and in vivo via its encapsulation in polymer-lipid hybrid nanoparticles coated with a polyethylene glycol shell. The nanoparticles are stable in serum, elicit low toxicity and enable high PTEN mRNA transfection in prostate cancer cells. Moreover, significant inhibition of tumour growth is achieved when delivered systemically in multiple mouse models of prostate cancer. We also show that the restoration of PTEN function in PTEN-null prostate cancer cells inhibits the phosphatidylinositol 3-kinase (PI3K)-AKT pathway and enhances apoptosis. Our findings provide proof-of-principle evidence of the restoration of mRNA-based tumour suppression in vivo.

Author Correction: Restoration of tumour-growth suppression in vivo via systemic nanoparticle-mediated delivery of PTEN mRNA.

The authors wish to add the following sentence into the 'Competing interests' section of this Article: "P.W.K. has investment interest in Context Therapeutics LLC, DRGT, Placon, Seer Biosciences and Tarveda Therapeutics, is a company board member for Context Therapeutics LLC, is a consultant and scientific advisory board member for BIND Biosciences, Inc., BN Immunotherapeutics, DRGT, GE Healthcare, Janssen, Metamark, New England Research Institutes, Inc., OncoCellMDX, Progenity, Sanofi, Seer Biosciences, Tarveda Therapeutics and Thermo Fisher, and serves on data safety monitoring boards for Genentech/Roche and Merck." This has now been included.

Biomaterials for mRNA delivery.

Messenger RNA (mRNA) has recently emerged with remarkable potential as an effective alternative to DNA-based therapies because of several unique advantages. mRNA does not require nuclear entry for transfection activity and has a negligible chance of integrating into the host genome which excludes the possibility of potentially detrimental genomic alternations. Chemical modification of mRNA has further enhanced its stability and decreased its activation of innate immune responses. Additionally, mRNA has been found to have rapid expression and predictable kinetics. Nevertheless, the ubiquitous application of mRNA remains challenging given its unfavorable attributes, such as large size, negative charge and susceptibility to enzymatic degradation. Further refinement of mRNA delivery modalities is therefore essential for its development as a therapeutic tool. This review provides an exclusive overview of current state-of-the-art biomaterials and nanotechnology platforms for mRNA delivery, and discusses future prospects to bring these exciting technologies into clinical practice.

The Contribution of Angiogenesis to the Process of Metastasis.

The role of angiogenesis in tumor growth has been studied continuously for over 45 years. It is now appreciated that angiogenesis is also essential for the dissemination and establishment of tumor metastases. In this review, we focus on the role of angiogenesis as a necessity for the escape of tumor cells into the bloodstream and for the establishment of metastatic colonies in secondary sites. We also discuss the role of tumor lymphangiogenesis as a means of dissemination of lymphatic metastases. Appropriate combination therapies may be used in the future to both prevent and treat metastatic disease through the rational use of antiangiogenic and antilymphangiogenic therapies in ways that are informed by the current and future work in the field.

Long-circulating siRNA nanoparticles for validating Prohibitin1-targeted non-small cell lung cancer treatment.

RNA interference (RNAi) represents a promising strategy for identification and validation of putative therapeutic targets and for treatment of a myriad of important human diseases including cancer. However, the effective systemic in vivo delivery of small interfering RNA (siRNA) to tumors remains a formidable challenge. Using a robust self-assembly strategy, we develop a unique nanoparticle (NP) platform composed of a solid polymer/cationic lipid hybrid core and a lipid-poly(ethylene glycol) (lipid-PEG) shell for systemic siRNA delivery. The new generation lipid-polymer hybrid NPs are small and uniform, and can efficiently encapsulate siRNA and control its sustained release. They exhibit long blood circulation (t1/2 ∼ 8 h), high tumor accumulation, effective gene silencing, and negligible in vivo side effects. With this RNAi NP, we delineate and validate the therapeutic role of Prohibitin1 (PHB1), a target protein that has not been systemically evaluated in vivo due to the lack of specific and effective inhibitors, in treating non-small cell lung cancer (NSCLC) as evidenced by the drastic inhibition of tumor growth upon PHB1 silencing. Human tissue microarray analysis also reveals that high PHB1 tumor expression is associated with poorer overall survival in patients with NSCLC, further suggesting PHB1 as a therapeutic target. We expect this long-circulating RNAi NP platform to be of high interest for validating potential cancer targets in vivo and for the development of new cancer therapies.

Identification of genes regulating migration and invasion using a new model of metastatic prostate cancer.

BACKGROUND: Understanding the complex, multistep process of metastasis remains a major challenge in cancer research. Metastasis models can reveal insights in tumor development and progression and provide tools to test new intervention strategies. METHODS: To develop a new cancer metastasis model, we used DU145 human prostate cancer cells and performed repeated rounds of orthotopic prostate injection and selection of subsequent lymph node metastases. Tumor growth, metastasis, cell migration and invasion were analyzed. Microarray analysis was used to identify cell migration- and cancer-related genes correlating with metastasis. Selected genes were silenced using siRNA, and their roles in cell migration and invasion were determined in transwell migration and Matrigel invasion assays. RESULTS: Our in vivo cycling strategy created cell lines with dramatically increased tumorigenesis and increased ability to colonize lymph nodes (DU145LN1-LN4). Prostate tumor xenografts displayed increased vascularization, enlarged podoplanin-positive lymphatic vessels and invasive margins. Microarray analysis revealed gene expression profiles that correlated with metastatic potential. Using gene network analysis we selected 3 significantly upregulated cell movement and cancer related genes for further analysis: EPCAM (epithelial cell adhesion molecule), ITGB4 (integrin ß4) and PLAU (urokinase-type plasminogen activator (uPA)). These genes all showed increased protein expression in the more metastatic DU145-LN4 cells compared to the parental DU145. SiRNA knockdown of EpCAM, integrin-ß4 or uPA all significantly reduced cell migration in DU145-LN4 cells. In contrast, only uPA siRNA inhibited cell invasion into Matrigel. This role of uPA in cell invasion was confirmed using the uPA inhibitors, amiloride and UK122. CONCLUSIONS: Our approach has identified genes required for the migration and invasion of metastatic tumor cells, and we propose that our new in vivo model system will be a powerful tool to interrogate the metastatic cascade in prostate cancer.

Hybrid lipid-polymer nanoparticles for sustained siRNA delivery and gene silencing.

The development of controlled-release nanoparticle (NP) technologies has great potential to further improve the therapeutic efficacy of RNA interference (RNAi), by prolonging the release of small interfering RNA (siRNA) for sustained, long-term gene silencing. Herein, we present an NP platform with sustained siRNA-release properties, which can be self-assembled using biodegradable and biocompatible polymers and lipids. The hybrid lipid-polymer NPs showed excellent silencing efficacy, and the temporal release of siRNA from the NPs continued for over one month. When tested on luciferase-expressed HeLa cells and A549 lung carcinoma cells after short-term transfection, the siRNA NPs showed greater sustained silencing activity than lipofectamine 2000-siRNA complexes. More importantly, the NP-mediated sustained silencing of prohibitin 1 (PHB1) generates more effective tumor cell growth inhibition in vitro and in vivo than the lipofectamine complexes. We expect that this sustained-release siRNA NP platform could be of interest in both fundamental biological studies and clinical applications. FROM THE CLINICAL EDITOR: Emerging gene silencing applications could be greatly enhanced by prolonging the release of siRNA for sustained gene silencing. This team of scientists presents a hybrid lipid-polymer nanoparticle platform that successfully accomplishes this goal, paving the way to future research studies and potential clinical applications.

Regulation of epithelial plasticity by miR-424 and miR-200 in a new prostate cancer metastasis model.

Using an in vivo cycling strategy, we selected metastatic cancer cells from the lymph nodes (LN) of mice bearing orthotopic DU145 human prostate tumors. Repeated rounds of metastatic selection (LN1-LN4) progressively increased the epithelial phenotype, resulting in a new model of tumor cell mesenchymal-epithelial transition (MET). DU145-LN4 showed increased cell-cell adhesions, higher expression of multiple epithelial markers, such as E-cadherin, EpCAM and cytokeratin 18, and reduced expression of mesenchymal markers such as vimentin. The MET in DU145-LN4 cells was accompanied by increased expression of the miR-200 family, and antimiRs to miR-200c and miR-141 induced an EMT. MET also correlated with the loss of miR-424. Ectopic transient and stable miR-424 expression induced EMT, with reduced epithelial marker expression and increased cell scattering. Our model provides evidence for spontaneous MET in vivo. We show that this cellular plasticity can be mediated through the combined action of miR-424 and the miR-200 family.

Interleukin-6 is required for pancreatic cancer progression by promoting MAPK signaling activation and oxidative stress resistance.

Pancreatic cancer, one of the deadliest human malignancies, is almost invariably associated with the presence of an oncogenic form of Kras. Mice expressing oncogenic Kras in the pancreas recapitulate the stepwise progression of the human disease. The inflammatory cytokine interleukin (IL)-6 is often expressed by multiple cell types within the tumor microenvironment. Here, we show that IL-6 is required for the maintenance and progression of pancreatic cancer precursor lesions. In fact, the lack of IL-6 completely ablates cancer progression even in presence of oncogenic Kras. Mechanistically, we show that IL-6 synergizes with oncogenic Kras to activate the reactive oxygen species detoxification program downstream of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling cascade. In addition, IL-6 regulates the inflammatory microenvironment of pancreatic cancer throughout its progression, providing several signals that are essential for carcinogenesis. Thus, IL-6 emerges as a key player at all stages of pancreatic carcinogenesis and a potential therapeutic target.

Cytoskeletal stiffness, friction, and fluidity of cancer cell lines with different metastatic potential.

We quantified mechanical properties of cancer cells differing in metastatic potential. These cells included normal and H-ras-transformed NIH3T3 fibroblast cells, normal and oncoprotein-overexpressing MCF10A breast cancer cells, and weakly and strongly metastatic cancer cell line pairs originating from human cancers of the skin (A375P and A375SM cells), kidney (SN12C and SN12PM6 cells), prostate (PC3M and PC3MLN4 cells), and bladder (253J and 253JB5 cells). Using magnetic twisting cytometry, cytoskeletal stiffness (g') and internal friction (g″) were measured over a wide frequency range. The dependencies of g' and g″ upon frequency were used to determine the power law exponent x which is a direct measure of cytoskeletal fluidity and quantifies where the cytoskeleton resides along the spectrum of solid-like (x = 1) to fluid-like (x = 2) states. Cytoskeletal fluidity x increased following transformation by H-ras oncogene expression in NIH3T3 cells, overexpression of ErbB2 and 14-3-3-ζ in MCF10A cells, and implantation and growth of PC3M and 253J cells in the prostate and bladder, respectively. Each of these perturbations that had previously been shown to enhance cancer cell motility and invasion are shown here to shift the cytoskeleton towards a more fluid-like state. In contrast, strongly metastatic A375SM and SN12PM6 cells that disseminate by lodging in the microcirculation of peripheral organs had smaller x than did their weakly metastatic cell line pairs A375P and SN12C, respectively. Thus, enhanced hematological dissemination was associated with decreased x and a shift towards a more solid-like cytoskeleton. Taken together, these results are consistent with the notion that adaptations known to enhance metastatic ability in cancer cell lines define a spectrum of fluid-like versus solid-like states, and the position of the cancer cell within this spectrum may be a determinant of cancer progression.

Insulin-like growth factors promote vasculogenesis in embryonic stem cells.

The ability of embryonic stem cells to differentiate into endothelium and form functional blood vessels has been well established and can potentially be harnessed for therapeutic angiogenesis. However, after almost two decades of investigation in this field, limited knowledge exists for directing endothelial differentiation. A better understanding of the cellular mechanisms regulating vasculogenesis is required for the development of embryonic stem cell-based models and therapies. In this study, we elucidated the mechanistic role of insulin-like growth factors (IGF1 and 2) and IGF receptors (IGFR1 and 2) in endothelial differentiation using an embryonic stem cell embryoid body model. Both IGF1 or IGF2 predisposed embryonic stem to differentiate towards a mesodermal lineage, the endothelial precursor germ layer, as well as increased the generation of significantly more endothelial cells at later stages. Inhibition of IGFR1 signaling using neutralizing antibody or a pharmacological inhibitor, picropodophyllin, significantly reduced IGF-induced mesoderm and endothelial precursor cell formation. We confirmed that IGF-IGFR1 signaling stabilizes HIF1α and leads to up-regulation of VEGF during vasculogenesis in embryoid bodies. Understanding the mechanisms that are critical for vasculogenesis in various models will bring us one step closer to enabling cell based therapies for neovascularization.

Knockdown of antizyme inhibitor decreases prostate tumor growth in vivo.

The endogenous protein antizyme inhibitor (AZI) is a potential oncogene which promotes cell growth by both inhibiting antizyme (AZ) activity and releasing ornithine decarboxylase (ODC) from AZ-mediated degradation. High levels of ODC and polyamines are associated with numerous types of neoplastic transformation, and the genomic region including AZI is frequently amplified in tumors of the ovary and prostate. To determine whether AZI functionally promotes prostate tumor growth, we made PC3M-LN4 (human) and AT6.1 (rat) cancer cell lines stably expressing shRNA to knockdown antizyme inhibitor 1 (AZI). AZI knockdown was confirmed by western blot, quantitative real-time PCR, and immunofluorescence. To examine the ability of these cells to form tumors in vivo, 1 × 10(6) cells were injected subcutaneously into nude mice either with (PC3M-LN4) or without (AT6.1) Matrigel. Tumor growth was measured two times per week by caliper. We found that cells in which AZI levels had been knocked down by shRNA formed significantly smaller tumors in vivo in both human and rat prostate cancer cell lines. These results suggest that not only does AZI promote tumor growth, but also that AZI may be a valid therapeutic target for cancer treatment.

Evidence of a role for antizyme and antizyme inhibitor as regulators of human cancer.

Antizyme and its endogenous antizyme inhibitor have recently emerged as prominent regulators of cell growth, transformation, centrosome duplication, and tumorigenesis. Antizyme was originally isolated as a negative modulator of the enzyme ornithine decarboxylase (ODC), an essential component of the polyamine biosynthetic pathway. Antizyme binds ODC and facilitates proteasomal ODC degradation. Antizyme also facilitates degradation of a set of cell cycle regulatory proteins, including cyclin D1, Smad1, and Aurora A kinase, as well as Mps1, a protein that regulates centrosome duplication. Antizyme has been reported to function as a tumor suppressor and to negatively regulate tumor cell proliferation and transformation. Antizyme inhibitor binds to antizyme and suppresses its known functions, leading to increased polyamine synthesis, increased cell proliferation, and increased transformation and tumorigenesis. Gene array studies show antizyme inhibitor to be amplified in cancers of the ovary, breast, and prostate. In this review, we summarize the current literature on the role of antizyme and antizyme inhibitor in cancer, discuss how the ratio of antizyme to antizyme inhibitor can influence tumor growth, and suggest strategies to target this axis for tumor prevention and treatment.